1. Field of the Invention
This invention relates to chemiluminescent N-alkylacridancarboxylate derivatives which allow the production of light (chemiluminescence) from the acridan by reaction with a peroxide and a peroxidase. This invention also relates to an improved method of generating light chemically (chemiluminescence) by the action of a peroxidase enzyme and an oxidant such as hydrogen peroxide with a group of N-alkylacridancarboxylate derivatives. The invention also relates to an improved method of enhancing the amount of chemiluminescence produced from this process by the use of specific substances. The invention also relates to the use of this method to detect the peroxidase enzyme. The invention also relates to the use of this method to detect hydrogen peroxide. Further, the invention relates to the use of the method to detect and quantitate various biological molecules. For example, the method may be used to detect haptens, antigens and antibodies by the technique of immunoassay, proteins by Western blotting, DNA and RNA by Southern and Northern blotting, respectively. The method may also be used to detect DNA in DNA sequencing applications. The method may additionally be used to detect enzymes which generate hydrogen peroxide such as glucose oxidase, glucose-6-phosphate dehydrogenase, galactose oxidase and the like as are generally known in the art.
2. Description of Related Art
The detection and quantitation of biological molecules has been accomplished historically with excellent sensitivity by the use of radiolabeled reporter molecules. Recently numerous non-radioactive methods have been developed to avoid the hazards and inconvenience posed by these materials. Methods based on enzyme-linked analytes offer the best sensitivity since the ability to catalytically turn over substrate to produce a detectable change achieves an amplification. Substrates which generate color, fluorescence or chemiluminescence have been developed, the latter achieving the best sensitivity.
Further increases in assay sensitivity will expand the range of utility of chemiluminescence-based methods by permitting the detection of analytes present in smaller quantities or reducing the amount of time and/or reagents required to perform the assay. A way to increase the speed and sensitivity of detection in an enzymatic chemiluminescent assay is through the use of substrates which generate light with a higher efficiency or for a greater length of time.
Among the enzymes used in enzyme-linked detection methods such as immunoassays, detection of oligonucleotides and nucleic acid hybridization techniques, the most extensively used to date has been horseradish peroxidase. Chemiluminescent reagents known in the art do not permit full advantage to be taken of the beneficial properties of this enzyme in analysis mainly due to sensitivity limitations. A reagent which permits the detection of lower amounts of enzyme is needed to enable the use of peroxidase conjugates in applications requiring ultrasensitive detection. Specifically, reagents are required which generate higher levels of chemiluminescence without an accompanying increase in the background or non-specific chemiluminescence. The increased chemiluminescence may be accomplished via either a higher maximum intensity or a longer duration than compounds known in the art.
a. Oxidation of acridan. Oxidation of acridan by benzoyl peroxide in aqueous solution produced chemiluminescence with very low efficiency (.phi..sub.CL =3.times.10.sup.-7) and a mixture of products including acridine (S. Steenken, Photochem. Photobiol., 11, 279-283 (1970)). N-Methylacridan is oxidized electrochemically to N-methylacridinium ion (P. Hapiot, J. Moiroux, J. M. Saveant, J. Am. Chem. Soc., 112(4), 1337-43 (1990); N. W. Koper, S. A. Jonker, J. W. Verhoeven, Recl. Trav. Chim. Pays-Bas, 104(11), 296-302 (1985)). Chemical oxidation of N-alkylacridan compounds has been performed with ferricyanide ion (A. Sinha, T. C. Bruice, J. Am. Chem. Soc., 106(23), 7291-2 (1984)), certain quinones (A. K. Colter, P. Plank, J. P. Bergsma, R. Lahti, A. A. Quesnel, A. G. Parsons, Can. J. Chem., 62(9), 1780-4 (1984)) and lithium nitrite (O. N. Chupakhin, I. M. Sosonkin, A. I. Matern, G. N. Strogov, Dokl. Akad. Nauk SSSR, 250(4), 875-7 (1980)). Oxidation of an N-alkylacridan derivative has been performed photochemically with or without a flavin compound as co-oxidant (W. R. Knappe, J. Pharm. Sci., 67(3), 318-20 (1978); G. A. Digenis, S. Shakshir, M. A. Miyamoto, H. B. Kostenbauer, J. Pharm. Sci., 65(2), 247-51 (1976)).
Aryl and alkyl esters of 10-methylacridan-9-carboxylic acid undergo autoxidation to N-methylacridone in dipolar aprotic solvents under strongly basic conditions to produce chemiluminescence (F. McCapra, Accts. Chem. Res., 9(6), 201-8 (1976); F. McCapra, M. Roth, D. Hysert, K. A. Zaklika in Chemiluminescence and Bioluminescence, Plenum Press, New York, 1973, pp. 313-321; F. McCapra, Prog. Org. Chem., 8, 231-277 (1971); F. McCapra, Pure Appl. Chem., 24, 611-629 (1970); U.S. Pat. No. 5,283,334 to McCapra). Chemiluminescence quantum yields ranged from 10.sup.-5 to 0.1 and were found to increase as the pKa of the phenol or alcohol leaving group decreased. Quantum yields in aqueous solution were significantly lower due a competing non-luminescent decomposition of an intermediate. Addition of the cationic surfactant CTAB increased the apparent light yield 130-fold by preventing a competing dark reaction.
Applicants' co-pending application Ser. No. 08/061,810 discloses the first use of an enzyme to oxidize substituted and unsubstituted N-alkylacridancarboxylic acid derivatives to generate chemiluminescence. In the presence of a peroxidase enzyme and a peroxide, N-alkylacridancarboxylate derivatives are efficiently oxidized to produce the N-alkylacridone and blue chemiluminescence.
b. Chemiluminescent oxidation of acridinium esters. The chemiluminescent oxidation of aliphatic and aromatic esters of N-alkylacridinium carboxylic acid by H.sub.2 O.sub.2 in alkaline solution is a well known reaction. The high chemiluminescence quantum yield approaching 0.1 has led to development of derivatives with pendant reactive groups for attachment to biological molecules. Numerous chemiluminescent immunoassays and oligonucleotide probe assays utilizing acridinium ester labels have been reported.
The use of acridinium esters (AE's), especially when labeled to a protein or oligonucleotide suffers from two disadvantages. The chief problem is limited hydrolytic stability. Acridinium ester conjugates decompose steadily at or slightly above room temperature. Depending on the substitution of the leaving group storage at -20.degree. C. may be required for extended storage.
A second disadvantage of acridinium esters is the tendency to add nucleophiles such as water at the 9-position to spontaneously form a pseudo-base intermediate which is non-luminescent and decomposes in a pH-dependent manner in a dark process. In practice the pH of solutions containing acridinium esters must be first lowered to reverse pseudo-base formation and then raised in the presence of H.sub.2 O.sub.2 to produce light.
Amides, thioesters and sulfonamides of N-alkylacridinium carboxylic acid have been shown to emit light when oxidized under these conditions (T. Kinkel, H. Lubbers, E. Schmidt, P. Molz, H. J. Skripczyk, J. Biolumin. Chemilumin., 4, 136-139, (1989), G. Zomer, J. F. C. Stavenuiter, Anal. Chim. Acta, 227, 11-19 (1989)). These modifications of the leaving group only partially improve the storage stability performance.
A more fundamental limitation to the use of acridinium esters as chemiluminescent labels lies in the fact that when used as direct labels, only up to at most about 10 molecules can be attached to a protein or oligonucleotide. Coupled with the quantum efficiency for producing a photon (.ltoreq.10%), an acridinium ester-labeled analyte can generate at most one photon of light. In contrast, enzyme-labeled analytes detected by a chemiluminescent reaction can potentially generate several orders of magnitude more light per analyte molecule detected by virtue of the catalytic action of the enzyme.
An attempt to increase the number of acridinium ester molecules associated with an analyte in an immunoassay was made by constructing an antibody-liposome conjugate wherein the liposome contained an unspecified number of AE's (S.-J. Law, T. Miller, U. Piran, C. Klukas, S. Chang, J. Unger, J. Biolumin. Chemilumin., 4, 88-98, (1989)). This method only produced a modest increase in signal over a comparable assay using directly labeled AE's.
c. Chemiluminescent Detection of Horseradish Peroxidase. Amino-substituted cyclic acylhydrazides such as luminol and isoluminol react with H.sub.2 O.sub.2 and a peroxidase enzyme catalyst (such as horseradish peroxidase, HRP) under basic conditions with emission of light. This reaction has been used as the basis for analytical methods for the detection of H.sub.2 O.sub.2 and for the peroxidase enzyme. An analog of luminol (8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4 (2H, 3H)dione) has been used in an enhanced chemiluminescent assay with HRP (M. Ii, H. Yoshida, Y. Aramaki, H. Masuya, T. Hada, M. Terada, M. Hatanaka, Y. Ichimori, Biochem. Biophys. Res. Comm., 193(2), 540-5 (1993)). Application of this compound in an immunoassay led to a two-fold lowering of the detection limit compared to detection using luminol. Another chemiluminescent compound oxidized by a peroxidase enzyme and a peroxide is a hydroxy-substituted phthalhydrazide (Akhavan-Tafti co-pending U.S. patent application No. 965,231, filed Oct. 23, 1992). Applicant's co-pending application Ser. No. 08/061,810 filed on May 17, 1993 discloses chemiluminescent N-alkylacridancarboxylic acid esters and sulfonimides which produce light upon reaction with a peroxide and a peroxidase for use in detecting peroxidase enzymes and in assays.
Numerous enhancers have also been employed in conjunction with the use of luminol to increase the intensity and duration of light emitted. These include benzothiazole derivatives such as D-luciferin, various phenolic compounds such as p-iodophenol and p-phenylphenol and aromatic amines (G. Thorpe, L. Kricka, in Bioluminescence and Chemiluminescence, New Perspectives, J. Scholmerich, et al, Eds., pp. 199-208 (1987)). For the purposes of the present discussion phenolic compounds are taken to mean hydroxylic aromatic compounds which will also include compounds such as 2-naphthol and 6-bromo-2-naphthol which are known to enhance other peroxidase reactions in addition to the aforementioned substituted hydroxyphenyl compounds. Other compounds which function as enhancers of the chemiluminescent oxidation of amino-substituted cyclic acylhydrazides by a peroxidase include 4-(4-hydroxyphenyl) thiazole (M. Ii, H. Yoshida, Y. Aramaki, H. Masuya, T. Hada, M. Terada, M. Hatanaka, Y. Ichimori, Biochem. Biophys. Res. Comm., 193 (2) , 540-5 (1993)), a group of compounds disclosed in U.S. Pat. No. 5,171,668 to Sugiyama, 2-hydroxy-9-fluorenone, and a group of hydroxy-substituted benzoxazole derivatives as disclosed in U.S. Pat. No. 5,206,149 to Oyama. The mechanism of oxidation of cyclic acylhydrazides by the combination of a peroxide and a peroxidase enzyme is very complex and remains the subject of intense debate (A. Lundin, L. Hallander, in Bioluminescence and Chemiluminescence, New Perspectives, J. Scholmerich, et al, Eds., pp. 555-558 (1987)); S. Vlasenko, A Arefyev, A. Klimov, B. Kim, E. Gorovits, A. Osipov, E. Gavrilova, A. Yegorov, J. Biolumin. Chemilumin. 4, 164-176 (1989)). This difficulty has hampered the development of new chemiluminescent reactions catalyzed by peroxidases.
d. Assays using HRP. The enzyme horseradish peroxidase has found widespread use in enzyme immunoassays and DNA hybridization assays with chemiluminescent detection using luminol or isoluminol as substrate (G. H. Thorpe, L. J. Kricka, S. B. Mosely, T. P. Whitehead Clin. Chem., 31, 1335 (1985), J. A. Matthews, A. Batki, C. Hynds, L. J. Kricka, Anal. Biochem., 151,205, (1985), P. Walsh, J. Varlaro, R. Reynolds, Nuc. Acids Res. 20,(19) 5061-5065 (1992)). Commercially available kits for conjugation of HRP with enhanced luminol chemiluminescent detection are available. Chemiluminescent assays using a peroxidase enzyme known in the art are not able to detect the lowest levels of certain analytes such as the thyroid hormone TSH, mainly due to the inability to detect the enzyme at extremely low levels. A chemiluminescent reagent which permits the detection of lower amounts of enzyme is needed for such assays.
e. Chemiluminescence Enhancement by Surfactants. Enhancement of chemiluminescent reactions using polymeric and monomeric surfactants is known in the art. Enhancement may occur by affecting the outcome of one or more steps e.g. by increasing the fluorescence quantum yield of the emitter, by increasing the percentage of product molecules produced in the excited state, by increasing the fraction of molecules undergoing the chemiluminescent reaction through inhibition of competing side reactions (McCapra Accts. Chem. Res., 9(6), 201-8 (1976)) or by promoting the action of an enzyme catalyst. No clear or consistent pattern exists concerning the effect of polymeric and monomeric surfactants on chemiluminescent reactions. It is impossible to predict which surfactant compounds, if any, may enhance the chemiluminescence from a particular process and can only be determined by substantial experimentation.
U.S. Pat. No. 5,145,772 to Voyta discloses enhancement of enzymatically generated chemiluminescence from 1,2-dioxetanes in the presence of polymeric compounds. Certain cationic polymer compounds were effective chemiluminescence enhancers; nonionic polymeric compounds were generally ineffective and the lone anionic polymer, Example 45, significantly decreased light emission.
U.S. Pat. No. 4,927,769 to Chang discloses enhancement by surfactants of the chemical oxidation of acridinium esters with alkaline hydrogen peroxide. These acridinium ester compounds are discrete from compounds of the present invention in that they react without the use of enzymes. Several of the tested surfactants (see Table 2 therein) provide only marginal enhancement.
A report on the effect of surfactants on the firefly luciferin-luciferase reaction (L. J. Kricka, M. DeLuca, Arch. Biochem. Biophys., 217, 674 (1983)) discloses enhancement of the light yield with nonionic surfactants by affecting the enzyme reactivity; a cationic surfactant totally extinguished light emission by inhibiting the enzyme.
A paper (T. Goto, H. Fukatsu, Tetrahedron. Lett., 4299 (1969)) teaches chemiluminescence enhancement of the chemical oxidation of Cypridina luciferin in the presence of nonionic and cationic but not anionic surfactants even though the fluorescence quantum yield of the emitter was increased in all three types of surfactants.
A paper (K. Sasamoto, Y. Ohkura, Chem. Pharm. Bull, 39(2), 411-6 (1991)) discloses enhancement by a cationic surfactant of chemiluminescence from chemical oxidation of a dialkylaminobenzofuranyl-substituted cyclic diacylhydrazide. An anionic surfactant was ineffective at enhancing the chemiluminescence, while a nonionic surfactant diminished light production.